1. Field of the Invention
This invention relates to novel methods for diagnosing, preventing or delaying the onset of, and treating urinary incontinence. The methods involve detecting and modulating the proteolysis of collagen in pelvic supporting tissue. More particularly, the methods of this invention relate to the differential expression of the matrix metalloproteinases (MMPs) known to degrade extracellular matrix collagen and the tissue inhibitors of metalloproteinases (TIMPs) which specifically inhibit MMP proteolytic activity. The invention also relates to a novel method of screening for specific modulators and inhibitors of pelvic collagen degradation.
2. State of the Art
Urinary incontinence and pelvic floor dysfunction are a major public health problem in the United States. A decade ago the National Institutes of Health estimated that at least 10 million American adults suffered from urinary incontinence (1). In addition, two-thirds of the burden of urinary incontinence is borne by women with prevalence rates ranging from 14% to 41% depending on the study population definition. Currently, American women can expect a 11.1% lifetime risk of undergoing surgery for incontinence or pelvic organ prolapse by the age of 80 and of those who do undergo surgery, close to 30% will require a repeat operation for recurrent urinary dysfunction (2), thus defining a growing area of disability for women for which no medical prophylaxis or therapy currently exists.
1. Collagen Content and Expression.
Collagen is composed of three polypeptide chains assembled in a triple helix, which is then supercoiled (3). Collagen forms the matrix of connective tissue and is made through very complex post-translational processing which ultimately requires crosslinking of the fascial collagen chains by pyridinoline in order to mature into a high tensile strength support structure (4). Over a dozen types of collagen have been identified, with types I and III being the main structural components of epithelial tissue (5), vaginal epithelium, and endopelvic fascia (6). Type I collagen is most abundant in the skin, tendons, ligaments, bones, and cornea where it comprises over 80% of the total collagen. Type II collagen is primarily present in cartilage, and type III collagen has the same distribution as does type I, but the ratio between the two varies. Type III collagen appears to be more abundant in the dermis during early fetal development and is thought to contribute to the elastic properties of the tissue (5).
Mechanical stability of the genito-urinary tract depends on intact, functional collagen fibers, which support the bladder neck, urethra, and pelvic organs. There are several reports in the literature that suggest that women with stress urinary incontinence, and perhaps those with prolapse as well (7), have lower levels of total collagen in vesico-vaginal fascia (8), abdominal skin, and round ligament (9) than control, continent women. Collagenous tissue from the vaginal wall is representative of tissue from the uterosacral and round ligaments (7) as well as tissue supporting the urethra, bladder, and bladder base (6). The content of type III collagen in perineal skin, uterosacral ligaments, and round ligaments of women with stress urinary incontinence was shown to be significantly reduced compared to that of women without SUI (6). In addition, there was a positive correlation between urethral pressure measurements and collagen content in abdominal skin as measured by hydroxyproline (10). Consistent with these in vivo observations, an in vitro study of fibroblasts cultured from skin biopsies of women with stress incontinence showed a 30% decrease in collagen accumulation compared to cultures from continent women (11).
Other studies have compared mRNA levels for specific collagen types (12), the pyridinoline crosslink content of collagen (13,14), collagen fiber size by transmission electron microscopy (12), ratios of type I/type III collagens (14), levels of type I collagen (15). However, such studies have yielded conflicting and/or inconclusive results.
2. Collagen Metabolism
Collagen is a metabolically active structure with constant turnover mediated through proteolysis by a group of enzymes termed matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of matrix metalloproteinase (TIMPs).
Matrix metalloproteinases (MMPs) are a family of structurally related proteins which degrade extracellular matrix and basement membrane components (16). They are produced as proenzymes and activated through proteolytic cleavage by other MMPs or plasmin. Interstitial collagens (types I, II, and III), the most abundant connective tissue proteins, are cleaved by the interstitial collagenases (MMP-1, MMP-8, and MMP-13) which specifically cleave native triple helical collagen at a single peptide bond in each a chain, yielding two fragments (17). This single site for cleavage (Gly775-Leu/Ile776) produces both three-quarter (TCA) and one-quarter (TCB) length collagen fragments (18) generating two neoepitopes: (COL2-3/4Cshort) on the carboxy-terminal end and COL2-1/4N1 on the amino-terminus. Specific antibodies to these carboxy- and amino-terminal neoepitopes have been utilized to document increased collagenase activity in cartilage from patients with osteoarthritis compared to nonarthritic cartilage (19) and excessive collagenase activity in human atheromatous plaques (20).
These one-quarter and three-quarter length collagen fragments are very susceptible to rapid gelatinase (MMP-2 and MMP-9) degradation to amino acids (21). Importantly, MMP-2 may also act on intact collagen degrading it to quarter length fragments similarly to MMP-1 (17). Thus, the interstitial collagenases and the gelatinases together are capable of completely degrading intact collagen fibers. As such, MMP-2 and MMP-9 may be critically important to loss of strength of fibrous collagen and, ultimately, with sufficient degradation, to loss of tissue strength.
In the extracellular matrix, the activity of MMPs is tightly regulated by a family of natural inhibitors, the tissue inhibitors of metalloproteinase (TIMPs). TIMP-1, TIMP-2, TIMP-3, and TIMP-4 have been described (22). TIMP-1 is a glycoprotein of molecular weight 28.5 kDa which binds stoichiometrically with MMP-9. TIMP-2 is a 21 kDa protein which binds to MMP-2. TIMP-3, a novel member of the TIMP family, has inhibitory activity against MMP-9 as well as stromelysin-1 and MMP-1. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix and it appears to be the most effective inhibitor of MMP-9 (23). The most recently discovered TIMP-4 appears primarily in cardiac tissue and may function to maintain extracellular matrix hemostasis (22). A single study addressed MMP levels and has shown significant increases in MMP-2 and MMP-9 in pelvic tissue from premenopausal women with prolapse, but not incontinence, when compared with normal women (7), implying increased metabolic turnover, and decreased mechanical strength in the tissue. There is no information about TIMP expression in pelvic tissue in incontinent women.
The MMP-2/TIMP-2 ratio has been found to correlate with the invasiveness of breast carcinomas, suggesting the potential utility of this ratio as an early prognostic tool to determine more reliably the aggressiveness of the breast cancer (24).
3. Modulators of MMP and/or TIMP Expression.
(a) GnRH Agonist: Trophoblast invasion is regulated in part by MMP-9 which can be inhibited by both TIMP-1 and TIMP-3 (25). We have shown that TIMP-1 and TIMP-3 mRNA expression in cultured human endometrial stromal cells and protein secretion into the medium were significantly decreased by GnRH agonist when compared to control cells. Moreover, the expression of TIMP-1 appeared to be affected to a much greater extent than that of TIMP-3 (26).
(b) Cytokines and Growth Factors: The cytokine IL-1xcex2 has also been shown to stimulate a two-fold increase in procollagenase production in pregnant Guinea pig cervix (27). Our laboratory has examined the ability of IL-1xcex2 and transforming growth factor-xcex2 (TGF-13) to regulate MMP-9, TIMP-1, and TIMP-3 mRNA expression in cultured human endometrial stromal cells (28). In these cultured endometrial stromal cells, TIMP-1 and TIMP-3 but not MMP-9 mRNA were expressed. However, incubation with IL-1xcex2 resulted in stimulation of MMP-9 expression and a decrease in both TIMP-1 and TIMP-3 mRNA expression in a dose-dependent maimer. Conversely, TGF-xcex2 augmented TIMP-1 and TIMP-3 mRNA expression suggesting that IL-1xcex2 and TGF-xcex2 may play opposing roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of both TIMPs and MMP-9 (28).
(c) Gonadal Steroids: Gonadal steroids have been shown both in vivo and in vitro to affect collagen status in women with SUI. Physiologically, estrogen has been shown to increase urethral pressure in postmenopausal women (29). In addition, approximately 50% of women with SUI reported subjective improvement in symptoms after estrogen treatment. For example, 6 of 11 patients who were given vaginal estrogen creme daily for 6 weeks were cured or improved significantly with that therapy (29). On the other hand, other studies have suggested that hypoestrogenism may simply affect the sensory threshold of the lower urinary tract, resulting in incontinence through a neural mechanism (30). Conflicting in vivo evidence suggests that mixed groups of pre- and postmenopausal women with SUI have a lower content of pelvic collagen than controls (6, 8, 9, 11), but estrogen-replaced postmenopausal women do not (15). There is no information on the effect of estrogens, in vitro, on collagen synthesis in fibroblast cultures from incontinent women.
In vitro studies of collagen metabolism as a function of gonadal steroids have primarily been performed in animal models. For example, in the pregnant guinea pig cervix, procollagenase activity doubled in the presence of 17xcex2-estradiol and estrone (27) while in cultured rabbit uterine cervical fibroblasts, treated with progesterone or 17xcex2-estradiol, levels of procollagenase in the culture medium declined (31). However, under the same conditions, the level of TIMPs in the culture media increased as a function of incubation with progesterone and 17xcex2-estradiol. The suppression of MMP production and increase in TIMP production by both steroids suggests that collagenolysis in rabbit uterine cervical fibroblasts is negatively regulated by steroid hormones which modulate the ratio of MMP/TIMP at a pretranslational level (31). In human endometrium, gene expression of matrix metalloproteinases during the menstrual cycle is repressed by progesterone which was confirmed by immunoprecipitation of MMPs in explant cultures (32). In other experiments with human fibroblast cultures (11, 33), the effect of gonadal steroids was not examined. However, confirmation of estrogen receptors (8, 34) and progesterone receptors (34) in female pelvic floor tissue is consistent with a gonadal steroid-mediated response and provides a rationale for estrogen treatment of women with uro-genital disorders (29).
A single report on androgens confirms the ability of intravenous dehydroepiandosterone sulfate (DHEAS), a weak androgen, to increase collagenase activity in the human cervix by over 150% (35). This experiment on human cervix was performed to determine the ability of DHEAS to induce cervical ripening but also suggests that there are androgen receptors in the human cervix which may be involved in modulation of collagenase activity
4. Reversal of Collagenolysis by TIMP Inhibitors
Excessive collagenolysis mediated by MMPs appears to accelerate human disease processes such as tumor invasion, joint destruction in arthritis, and vascular aneurysm formation. Several studies have examined the ability of MMP inhibitors to reverse these pathologies. For example, BB-94 (Batimastat), a specific inhibitor of MMPs, was evaluated for its ability to control aneurysm growth in rat aorta. Rats treated with this inhibitor showed significantly less aneurysmal dilatation than did control rats (36). Others have shown that synthetic, pharmaceutically developed metalloproteinase inhibitors that mimic TIMP-2 but not TIMP-1 activity can inhibit shedding of TNF-xcex1 receptors in the human colon adenocarcinoma cell line (37). One of these drugs, Marimastat, has been used in a Phase I study to evaluate treatment of inpatients with advanced lung carcinoma. Marimastat was found to be well absorbed from the GI tract reaching plasma levels substantially higher than those required for MMP inhibition in vitro (38). Other MMP inhibitors have been developed and tested as potential therapeutic agents in the treatment of abdominal aortic aneurysm For example, RS 132,908, promoted preservation of aortic elastin and an enhanced fibrotic response within the aortic wall in male rats (39). RS 113,456 has been shown in vivo in male rats to diminish flow mediated arterial enlargement through MMP inhibition. This drug inhibits MMP-2 and MMP-9 with a KI in the human of 0.054 and 0.065 (nmol/L), respectively, thus providing an order of magnitude greater inhibition of MMP-2 and MMP-9 than for other MMPs (40).
In view of the high prevalence rates of urinary incontinence among older women, and the absence of a medical therapy for this disorder, there is a continuing need for methods that are capable of detecting predisposition to development of urinary dysfunction leading to stress urinary incontinence in women. As well, there is a need for methods of screening for therapeutic agents that are capable of reversing the pathophysiologic changes that appear to be associated with collagen degradation in pelvic tissue of women as they age.
The present invention satisfies the above needs by providing methods for diagnosing and treating urinary incontinence by detecting and reducing proteolysis of collagen in pelvic supporting tissue. The diagnostic methods are useful in detecting predisposition to development of urinary incontinence such that prophylactic treatment can be initiated to prevent the onset of symptoms.
One preferred embodiment of the invention is a method that sensitively reflects variations in collagen turnover in pelvic support tissue and can be used to detect predisposition to development of urinary incontinence and other conditions that are related to the weakening of mechanical stability of support tissues of the genitourinary tract (e.g., conditions resulting from genetic predisposition, hormonal imbalance, trauma and others). Another preferred embodiment is a method for diagnosing urinary incontinence in a subject based on a determination of the rate or degree of proteolysis of collagen in pelvic supporting tissue. The method is based on the inventors"" experimental findings that expression levels of MMPs and/or TIMPs are outside xe2x80x9cnormalxe2x80x9d ranges, and that MMP/TIMP expression ratios are elevated in pelvic supporting tissue of women with urinary incontinence, as compared to women with normal urinary function.
According to one embodiment, methods of the present invention involve determining the level of at least one MMP in pelvic supporting tissue in the subject, determining the level of at least one TIMP in pelvic supporting tissue in the subject; calculating a ratio of MMP/TIMP levels in the subject; and comparing the ratio to a predetermined indicator. The predetermined indicator is typically based on statistical assessment of MMP/TIMP expression ratios obtained from subjects with normal urinary function and subjects with actual or incipient urinary incontinence. Ratios can be determined from quantitative measurements of mRNA and protein levels or activities.
According to another embodiment, methods of the present invention involve determining collagen degradation and/or content in pelvic supporting tissue in a subject and comparing the collagen degradation and/or content to a predetermined indicator. Preferably, collagen degradation assays will measure the amount of initial cleavage products of collagen breakdown by MMPs (e.g., the carboxy-terminal neoepitope COL2-3/4 Cshort).
In another embodiment, the MMP/TIMP ratio method is used to determine whether a patient may benefit by treatment with one or more agents for modulating the expression or activity of MMPs and TIMPs (e.g., gonadal steroids, cytokines, growth factors, MMP inhibitors, TIMP analogs or derivatives) and/or collagenases.
The present invention also provides a diagnostic kit for assessing whether levels or activities of MMPs and TIMPs in a biological sample from an individual with symptoms of urinary incontinence are abnormal. The biological sample may be a bodily fluid, a cell or tissue sample or an extract or lysate thereof, or a cultured tissue. In one embodiment, the kit comprises a test for the activity or level of one or more MMPs, a test for the level of one or more TIMPs in the sample, and a predetermined indicator of normal and abnormal values for the tests. The predetermined indicator is an empirically determined value or range of values that is typically determined from test measurements on groups of normal subjects and subjects with urinary incontinence (or a predisposition for urinary incontinence). The test may be quantitative, e.g., for use in a clinical laboratory setting, or qualitative (e.g., detecting TIMP or MMP only when present in the sample at a certain predetermined cut-off level), e.g., for home use. Positive and negative controls may also be included in the kit. In another embodiment, the kit comprises a test for collagenase activity. Various technologies for home testing can be used in the practice of this embodiment. For example, tests are available that utilize specific antibodies or enzyme substrates bound to a substratum for detecting a protein level (or activity) or range of levels (or activities) in a predetermined volume of biological sample. The technologies that form the basis for these tests are well known to those skilled in clinical laboratory medicine. Any test that distinguishes between normal and abnormal levels can be used, provided that it is convenient to perform and is specific for measurement of the desired parameters.
Various procedures for quantitating protein levels, enzymatic activities and/or mRNAs are illustrated in the Examples below. Of course, it is recognized that other useful measurement techniques are well-known to biochemists, immunobiologists and molecular biologists, and can be substituted for the procedures described herein.
Screening assays for identifying therapeutic agents that modulate proteolysis of collagen in pelvic supporting tissue are also provided by the present invention. These assays may be carried out with cell-free preparations (e.g., cell extracts, isolated collagenases, MMPs, TIMPs), isolated tissues and cell preparations, or cultured cells. It is anticipated that for some purposes (e.g., screening of libraries of chemical compounds and biotherapeutics), a first-stage screen that uses cell-free preparations will be employed to identify putative therapeutic modulators. Following this, one or more second-stage cell-based screens can be used to assess efficacy.
In general, the screening assays are in vitro assays that employ pelvic support tissue explants, cultures of cells derived from pelvic support tissues, or cultures of well-characterized cells that simulate the aberrant collagen turnover of pelvic support tissues from subjects with urinary incontinence. The screening assays involve measuring the activity of MMPs, or the relative protein levels of MMPs and TIMPs in pelvic support tissues or cells before and after treatment with at least one candidate therapeutic agent, and comparing the before and after activities or protein levels.
In another embodiment, a candidate therapeutic agent is screened for its effect on collagen proteolysis in pelvic support tissue explants or cultured cells, e.g., by quantitating the carboxy-terminal neoepitope produced by collagen cleavage. This direct measurement of collagenase activity may be carried out in conjunction with measurements of activity of MMPs or relative protein level of MMPs and TIMPs, as described above, to identify agents that selectively modulate collagenase activity and agents that have multiple effects on collagen turnover.
Therapeutic agents discovered with the use of the screening assays disclosed herein are also intended to be within the scope of this invention.
The present invention also provides methods of treating urinary incontinence, preferably in a mammal, and most preferably in a human. These methods encompass both prophylactic therapy (i.e., preventing or delaying the onset of urinary incontinence in predisposed subjects), and treatment of actual urinary incontinence. Each of the therapies is directed to reducing or proteolysis of collagen in pelvic supporting tissue. Preferred methods for reducing proteolysis of collagen in pelvic supporting tissue include: modulatin at least one of the level and activity of MMPs in the pelvic supporting tissue; modulating at least one of the level and activity of TIMPs in pelvic supporting tissue; and administering an agent that reduces or inhibits proteolysis of collagen in pelvic supporting tissue (e.g., gonadal steroids, growth factors, cytokines, and combinations thereof, TIMPs, TIMP analogs or derivatives).
It is preferred that MMP modulators used for the treatment of urinary incontinence include modulators of MMP-1, MMP-2 and MMP-9 which are known to be involved in collagen breakdown in pelvic supporting tissue. These modulators may be naturally-occurring tissue inhibitors of MMP activity (e.g., TIMP-1, TIMP-2, TIMP-3, TIMP-4) and synthetic derivatives and analogs thereof. Examples of such inhibitors include Batimastat (British Biotechnology Ltd., see EP-A-276436, and reference 36 disclosed herein), Marimastat (38) and RS 113,456 (Roche). Many other specific MMP inhibitors have been developed by pharmaceutical companies, including collagenase inhibitors, and are described in the patent literature. See, e.g., U.S. Pat. No. 6,127,427 and other patents cited therein. Inhibitors have also been developed that are active against several different MMPs (e.g., GM6001 (Galardin). It is expected that other useful inhibitors of MMP activity, such as peptides and peptidomimetics, will be identified using the screening assays of this invention.
Presently preferred modulators of MMP and TIMP expression include gonadal steroids, growth factors, cytokines and combinations of these.
Oligonucleotides directed against the sense strands of MMPs and TIMPs (i.e., antisense molecules) may also be of use in modulating the expression levels of MMPs and TIMPs. The design and administration of such antisense oligonucleotides is well known in the art.
The therapeutic compounds of the invention are usually administered in the form of pharmaceutical compositions. This invention therefore provides pharmaceutical compositions which contain, as the active ingredient, one or more compounds that modulate the proteolysis of collagen in pelvic supporting tissues and one or more pharmaceutically acceptable excipients, carriers, diluents, permeation enhancers, solubilizers and adjuvants. One or more compounds may be administered alone or in combination with other therapeutic agents, carriers, adjuvants, permeation enhancers, and the like. The compounds may be formulated using conventional techniques such as those described in Remington""s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985) and xe2x80x9cModern Pharmaceuticsxe2x80x9d, Marcel Dekker, Inc. 3rd Ed. (G. S. Banker and C. T. Rhodes, Eds. Pharmaceutically acceptable salts of the active agents (e.g., acid addition salts) may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, e.g., by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).
The compounds of the invention may be administered by any of the accepted modes of administration of agents having similar utilities and/or pharmacokinetics, for example, by oral, topical, or by parenteral routes (e.g., intradermal, intravenous, subcutaneous, intramuscular), intra-articular, intraspinal, epidural, rectal, vaginal, or transdermal/transmucosal routes. The most suitable route will depend on the nature and severity of the condition being treated. Oral delivery and sustained local delivery to pelvic supporting tissue are presently preferred routes for the compounds of this invention. In making the compositions of this invention, the active ingredient is customarily diluted by an excipient. Some examples of suitable excipients for oral formulations include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, PEG, polyvinylpyrrolidone, cellulose, water, sterile saline, syrup, and methyl cellulose. The formulations can additionally include: permeation enhancers, pH regulators, buffers, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
Alternatively, the compounds of this invention, particularly antisense molecules, peptides and other biotherapeutic modulators of MMP and TIMP expression, may be solubilized and encapsulated (e.g., in a lipo some or a biodegradable polymer) (52).
For topical use, the compositions can be in the form of emulsions, creams, jelly, solutions, ointments containing, for example, up to 5% by weight of the active compound. For parenteral administration, the compositions can be in the form of sterile injectable solutions and sterile packaged powders.
The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations.
Another preferred formulation for use in the methods of the present invention employs transdermal delivery devices (xe2x80x9cpatchesxe2x80x9d). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art.
The compositions are preferably formulated in a unit dosage form. The term xe2x80x9cunit dosage formsxe2x80x9d refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e.g., an ampoule).
The compounds of the present invention, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount. Typical dosage levels are in the range of about 0.1 mg to about 1,000 mg per day. A dosage in the range of about 0.01 to about 100 mg per kg of body weight per day is preferred. It will be understood, however, that the amount of the compound actually administered will be determined by a clinician of ordinary skill in the art, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient""s symptoms, and the like. The compounds of this invention are intended for medical use in humans and nonhuman mammals with analogous disorders and conditions.